Release of rhodanese from Pseudomonas aeruginosa by cold shock and its localization within the cell

1979 ◽  
Vol 25 (3) ◽  
pp. 340-351 ◽  
Author(s):  
R. W. Ryan ◽  
M. P. Gourlie ◽  
R. C. Tilton
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Outer Membrane ◽  
Electron Micrographs ◽  
Cold Shock ◽  
Cytoplasmic Membrane ◽  
Lactic Dehydrogenase ◽  
Periplasmic Space ◽  
Whole Cells ◽  
Periplasmic Enzyme

Whole cells of Pseudomonas aeruginosa possess rhodanese activity. The enzyme can be released by rapidly resuspending the cells in cold Tris–HCl buffer. Approximately 95% of the rhodanese activity is released by cold shock. Release of the enzyme can be inhibited either by preincubating the cells with Mg2+ or by incorporating Mg2+ into the shocking buffer. The effect of Mg2+ can be reversed by washing the cells twice with buffer prior to cold shock. While rhodanese can be released from P. aeruginosa by cold shock, lactic dehydrogenase, a cytoplasmic enzyme, remains within the cell. Diazo-7-amino-1,3-naphthalenedisulfonic acid, a compound which does not penetrate the cytoplasmic membrane, completely inactivated rhodanese and alkaline phosphatase, a periplasmic enzyme, whereas lactic dehydrogenase retained its full activity. These data suggest that rhodanese in P. aeruginosa, like alkaline phosphatase, is located distal to the cytoplasmic membrane in the periplasmic space. Electron micrographs also show that portions of the lipopolysaccharide outer membrane are shed from the cell during cold shock, while cells preincubated with Mg2+ did not release segments of their outer membrane.

Alkaline phosphatase of Pseudomonas aeruginosa: the mechanism of secretion and release of the enzyme from whole cells

1973 ◽  
Vol 19 (11) ◽  
pp. 1407-1415 ◽  
Author(s):  
J. M. Ingram ◽  
K. -J. Cheng ◽  
J. W. Costerton
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Cell Wall ◽  
Culture Filtrate ◽  
Outer Wall ◽  
Outer Cell ◽  
Periplasmic Space ◽  
Electron Microscopic ◽  
Whole Cells ◽  
Variable Quantity

The release of alkaline phosphatase from whole cells of Pseudomonas aeruginosa as a function of the MgCl2 concentration is proportional to the release of lipopolysaccharide from the cells. Cells grown under conditions where APase is almost completely secreted to the culture filtrate, i.e. growth at pH 7.6, also secrete lipopolysaccharide. Twenty percent sucrose releases a variable quantity of whole cell phosphatase. Localization of this portion of enzyme by biochemical and electron-microscopic techniques showed that it is located on the cell surface exterior to the outer tripartite layer. Phosphatase, which is not released by sucrose, but which is released by MgCl2, is located in the periplasmic space. Phosphatase is located in three areas; the culture filtrate, the outer cell wall surface, and the periplasmic space. The results suggest that A Pase is associated with, and bound to, a cell wall fraction which contains lipopolysaccharide and that the enzyme is "transported" through the outer wall in complex with this fraction. Liberation of the complex from the outer wall may be accomplished by the mechanical shearing forces developed during growth or during the sucrose suspension procedure.

scholarly journals Pyoverdine-Mediated Iron Uptake in Pseudomonas aeruginosa: the Tat System Is Required for PvdN but Not for FpvA Transport

2006 ◽  
Vol 188 (9) ◽  
pp. 3317-3323 ◽  
Author(s):  
Romé Voulhoux ◽  
Alain Filloux ◽  
Isabelle J. Schalk
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Iron Uptake ◽  
Cytoplasmic Membrane ◽  
Signal Sequence ◽  
Iron Release ◽  
Outer Membrane Receptor ◽  
Uptake Pathway ◽  
Uptake Process ◽  
Tat System

ABSTRACT Under iron-limiting conditions, Pseudomonas aeruginosa PAO1 secretes a fluorescent siderophore called pyoverdine (Pvd). After chelating iron, this ferric siderophore is transported back into the cells via the outer membrane receptor FpvA. The Pvd-dependent iron uptake pathway requires several essential genes involved in both the synthesis of Pvd and the uptake of ferric Pvd inside the cell. A previous study describing the global phenotype of a tat-deficient P. aeruginosa strain showed that the defect in Pvd-mediated iron uptake was due to the Tat-dependent export of proteins involved in Pvd biogenesis and ferric Pvd uptake (U. Ochsner, A. Snyder, A. I. Vasil, and M. L. Vasil, Proc. Natl. Acad. Sci. USA 99:8312-8317, 2002). Using biochemical and biophysical tools, we showed that despite its predicted Tat signal sequence, FpvA is correctly located in the outer membrane of a tat mutant and is fully functional for all steps of the iron uptake process (ferric Pvd uptake and recycling of Pvd on FpvA after iron release). However, in the tat mutant, no Pvd was produced. This suggested that a key element in the Pvd biogenesis pathway must be exported to the periplasm by the Tat pathway. We located PvdN, a still unknown but essential component in Pvd biogenesis, at the periplasmic side of the cytoplasmic membrane and showed that its export is Tat dependent. Our results further support the idea that a critical step of the Pvd biogenesis pathway involving PvdN occurs at the periplasmic side of the cytoplasmic membrane.

In vitro studies of an alkaline phosphatase – cell wall complex from Pseudomonas aeruginosa

1975 ◽  
Vol 21 (1) ◽  
pp. 9-16 ◽  
Author(s):  
D. F. Day ◽  
J. M. Ingram
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Cell Wall ◽  
Complex Formation ◽  
Wall Material ◽  
Outer Cell ◽  
Whole Cells ◽  
Wall Components ◽  
Outer Cell Wall

Alkaline phosphatase (APase) of Pseudomonas aeruginosa exists primarily in the periplasmic region of the cell, i.e., between the cytoplasmic membrane and the outer tripartite layer. The enzyme is also found in the culture filtrate or associated with the outer layer of the cell wall. APase forms a complex with released outer cell wall material, and lipopolysaccharide (LPS) is associated with the complex. Since the enzyme was purified to homogeneity, it became desirable to determine whether complex formation with LPS, or the outer cell wall, affected any properties of the purified phosphatase. The ratio of activities of purified APase with p-nitrophenylphosphate and β-glycerolphosphate as substrates is about 4:1. The ratio of activities with enzyme complexed with LPS is about 1:1. The energy of activation of sucrose or magnesium released enzyme is 9500 cal/mol whereas the values for purified enzyme plus LPS, purified enzyme, purified enzyme plus phosphatidylethanolamine (PE), and purified enzyme plus LPS plus PE range from 3400 to 8700 cal/mol. These changes occur in the physiological temperature range, 27 to 39C, of this organism. Sucrose-released enzyme in the presence of substrate is inactivated at 47C whereas pure enzyme plus substrate is affected at 41C. The addition of LPS, PE, or a combination of both increases the temperature of inactivation from 45 to 51C. The results suggest that certain properties of the purified enzyme differ from those of the enzyme released from whole cells by either sucrose or magnesium resuspension. The addition of cell wall components such as LPS and PE to purified APase restores these properties. The evidence suggests that artificial complex formation changes the environment of the enzyme protein such that the environment now resembles that which exists within the whole cell wall.

Localization of L-asparaginase in Pseudomonas aeruginosa

1971 ◽  
Vol 17 (8) ◽  
pp. 1025-1028 ◽  
Author(s):  
D. F. Day ◽  
J. M. Ingram
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lactic Acid ◽  
Cytoplasmic Membrane ◽  
Nadh Oxidase ◽  
Disulfonic Acid ◽  
Lactic Acid Dehydrogenase ◽  
Whole Cells ◽  
Acid Dehydrogenase ◽  
Membrane Enzyme ◽  
Asparaginase Activity

Whole cells of Pseudomonas aeruginosa possess L-asparaginase activity. The enzyme is released by suspension in 0.2 M MgCl2 and resuspension in 0.1 M Tris buffer, pH 8.4. Under these conditions neither the plasma membrane enzyme, NADH oxidase, nor the scluble enzyme, lactic acid dehydrogenase, are released. The L-asparaginase and the periplasmic alkaline phosphatase are completely inactivated by treatment of whole cells with diazonaphthalene–disulfonic acid, a reagent which does not penetrate the cytoplasmic membrane. Under the conditions used no inactivation of NADH oxidase or lactic acid dehydrogenase was observed. L-Asparaginase is also released by converting whole cells to spheroplasts. The results suggest that L-asparaginase of P. aeruginosa is located exterior to the cytoplasmic membrane, possibly in the periplasmic space.

Adaptive resistance to polymyxin in Pseudomonas aeruginosa due to an outer membrane impermeability mechanism

1982 ◽  
Vol 28 (7) ◽  
pp. 830-840 ◽  
Author(s):  
H. E. Gilleland Jr. ◽  
Linda B. Farley
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Resistant Strain ◽  
Cytoplasmic Membrane ◽  
Osmotic Shock ◽  
Membrane Repair ◽  
Permeability Characteristics ◽  
Minimal Inhibitory Concentrations ◽  
Adaptive Resistance ◽  
Resistant Strains

The isolated outer membrane from cells of a Pseudomonas aeruginosa strain exhibiting adaptive resistance to polymyxin was not affected by polymyxin treatment, as monitored by electron microscopy of negatively stained preparations. This was in sharp contrast with extensive disruption by polymyxin of the outer membranes of the parent polymyxin-sensitive strain and the resistant strain following reversion to greater polymyxin sensitivity. The isolated cytoplasmic membrane of the polymyxin-resistant strain, on the other hand, remained sensitive to the disruptive effects of polymyxin treatment. The permeability characteristics of the resistant strains appeared to be altered, as indicated by differences in minimal inhibitory concentrations for a variety of antibiotics between the polymyxin-sensitive and polymyxin-resistant strains. No evidence was found for a polymyxin-inactivating enzyme in osmotic shock fluid from the polymyxin-resistant strain. No evidence for a cytoplasmic membrane repair mechanism was found in the polymyxin-resistant strain. These observations suggest that the mechanism of adaptive polymyxin resistance in this model system is the alteration of the outer membrane so that it excludes polymyxin from reaching the still sensitive cytoplasmic membrane.

Glutaraldehyde reactions with alkaline phosphatase of Pseudomonas aeruginosa

1981 ◽  
Vol 27 (5) ◽  
pp. 531-535 ◽  
Author(s):  
D. F. Day ◽  
Jordan M. Ingram
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Outer Membrane ◽  
Comparative Studies ◽  
Cell Envelope ◽  
Cross Linking ◽  
Cytochemical Localization ◽  
Whole Cell ◽  
Kinetics Of

Glutaraldehyde, the biological fixative of choice in the cytochemical localization of the phosphatases, was investigated for its effects on Pseudomonas aeruginosa alkaline phosphatase. Comparative studies on the inactivation of alkaline phosphatase by glutaraldehyde showed significant differences when the purified protein was compared with whole, cell-bound enzyme.The effects of the reagent on the kinetics of the purified enzyme were studied and some conclusions drawn as to the mode of inactivation. The reaction of glutaraldehyde with the cell envelope of P. aeruginosa was also investigated, and it was found not to modify the extraction of lipopolysaccharides from the outer membrane. This study emphasizes the care that must be taken to interpret data, cytochemical or otherwise, obtained when glutaraldehyde is used as a fixative or cross-linking reagent.

Alkaline phosphatase localization and spheroplast formation of Pseudomonas aeruginosa

1970 ◽  
Vol 16 (12) ◽  
pp. 1319-1324 ◽  
Author(s):  
K. -J. Cheng ◽  
J. M. Ingram ◽  
J. W. Costerton
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Cell Wall ◽  
Electron Microscopic ◽  
Erroneous Conclusion ◽  
Lead Salts ◽  
Phosphatase System ◽  
Low Levels ◽  
Cell Wall Deposition ◽  
Periplasmic Enzyme

Pseudomonas aeruginosa cells grown in the presence of low levels of inorganic phosphate contain an inducible alkaline phosphatase system. The enzyme has been localized by electron microscopic techniques in the region between the cytoplasmic membrane and the tripartite layer of the cell wall, i.e. the periplasmic space. No deposits of lead salts are observed upon examination of either uninduced cells or cells in which the enzyme has been completely removed by 0.2 M magnesium washing. Samples of cells which were treated with glutaraldehyde before enzyme localization studies show cell wall deposition of lead salts which could give rise to the erroneous conclusion that the alkaline phosphatase was located in the tripartite layer. Cytochemical and biochemical studies are presented which show that discontinuities within the cell wall are insufficient to account for the release of this periplasmic enzyme and that dissociation by a divalent metal, increased pH, or both is required. As a consequence of this study it was possible to prepare true spheroplasts of P. aeruginosa.

Analysis by flow cytometry of surface-exposed epitopes of outer membrane protein F ofPseudomonas aeruginosa

1996 ◽  
Vol 42 (8) ◽  
pp. 859-862 ◽  
Author(s):  
Eileen E. Hughes ◽  
H. E. Gilleland Jr. ◽  
Janice M. Matthews-Greer
Keyword(s):  
Flow Cytometry ◽  
Pseudomonas Aeruginosa ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Median Percentage ◽  
Peptide Conjugates ◽  
Whole Cells ◽  
Protein F ◽  
Outer Membrane Protein F

Antisera were produced in mice immunized with 18 synthetic peptide conjugates representing various regions throughout the length of the outer membrane protein F molecule of Pseudomonas aeruginosa and analysed by flow cytometry to identify those antisera capable of binding to the surface of whole cells of P. aeruginosa. Antibodies to peptides 9, 18, 10, and 4 were significantly cell-surface reactive. The maximum median percentage of antibody-binding cells in this assay was 36.6%. Over six different determinations, peptide 9 antisera binding to the cells ranged from 16.9 to 57.0% of the cell population. We propose that the surface accessibility of protein F epitopes varies during the cell cycle.Key words: surface-exposed epitopes, outer membrane protein F, Pseudomonas aeruginosa, flow cytometry.

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